The present invention relates to a polysaccharide peptide conjugate and a process for making it. In a particular embodiment of the invention, the conjugate uses bacterial or fungal polysaccharides and thus can be useful for vaccine purposes.
Polysaccharides constitute a broad family of polymeric molecules which are useful in various technical fields. In some cases, they require to be coupled to a polypeptide, e.g. protein or peptide. For example, polysaccharides are used in diagnosis or purification technics as a matrix medium for peptide reagents. Non-immunogenic polysaccharides such as dextran, are also useful to present small peptides, to the immune system as described in EP 326 111. Indeed, peptides have to be linked to protein carriers or have to be administered with an adjuvant, the most commonly used adjuvants being aluminium compounds. However, small peptides mixed, adsorbed or precipitated with these adjuvants may be hindered by the aluminium gel and therefore, not available to the immune system. To overcome this problem, EP 326 111 teaches that peptides may be conjugated to non-immunogenic polysaccharides. Such conjugates, in the presence of aluminium compounds, are able to elicit an immune response against the peptide moiety.
In the vaccinal field, it is also highly interesting to conjugate polypeptides e.g., peptide or protein, to immunogenic polysaccharides, this once pursuing an immune response to the polysaccharides. Indeed, capsule and cell wall of bacteria (and also cell wall of fungi) are essentially constituted by polysaccharides composed of very specific repeat units that bear epitope motives that are usually not found in mammals and can mediate immunogenicity. Therefore, polysaccharides e.g., capsular polysaccharides have been already used as vaccines against bacterial diseases such as meningitis, pneumonia and typhoid fever.
However, there is a major problem when using polysaccharides as vaccines. Although they have been proven immunogenic i.e., in other words, they elicit an immune response when administered as such to a mammal, even if this response may be poor, they are specific in that they belong to the small number of antigens that are able to induce B-cells production without help from T-cells. Accordingly, they are called T-independent.
The immune response induced by T-independent antigens is characterized by a number of features, among which:
(i) The primary response is weaker and earlier than the response to T-dependent antigens;
(ii) The antibody response does not mature into high IgG production, with affinity increase, as observed with T-dependent antigens;
(iii) The immune memory corresponding to T-independent antigens is poor and thus, as the immune memory is the key of the secondary immune response that constitutes the basis of the vaccination principle, a T-independent antigen is a poor antigen for inducing a long term protective immune response; and
(iv) Infants are unable to respond to polysaccharides before one or two years of age.
In order to induce a secondary immune response, T-independent antigens require to be covalently coupled to a carrier protein such as diphtheria or tetanus toxin, which give the antigen the T-dependent character. The conjugate thereof may then be complemented with an adjuvant such as an aluminium compound or the complete or incomplete Freund""s adjuvant (these two latter, exclusively for use in mammals other than humans), so that the immune response is enhanced (adjuvant effect).
By the term xe2x80x9ccarrierxe2x80x9d is meant a molecule that, when covalently linked to an antigen e.g. a polysaccharide, is capable of promoting a T-dependent response to the antigen. Such a response is shown upon a vaccination scheme comprising at least two injections of the antigen-carrier conjugate, at days, weeks or months apart (priming and booster). Upon the first infection (priming), a weak antibody response is shown, while upon the booster injection, the antibody response is elicited at a high level. Such a magnified response is not to be seen with the negative control constituted by the unconjugated antigen.
Various conjugation methods are already available in the art. Polysaccharide functional groups that are commonly involved, may be amino, carboxyl or hydroxyl groups located along the chain or aldehyde groups either terminal or along the chain. Polypeptide functional groups that are usually involved, may be amino or carboxyl groups, terminal or present on the amino acid side chains or even thiol groups.
In a general manner, polysaccharide conjugates may exibit three types of structure depending upon the location of functional groups of both polysaccharide (either along the chain or at the end) and carrier, that are involved in the linkage. These types of structure are called for ease of description, xe2x80x9cSunxe2x80x9d or xe2x80x9cEarxe2x80x9d, xe2x80x9cRakexe2x80x9d and xe2x80x9cLatticexe2x80x9d types. They are illustrated in FIG. 1, wherein (A), (B) and (C) respectively stand for xe2x80x9cSunxe2x80x9d (neoglycoconjugate), xe2x80x9cRakexe2x80x9d and xe2x80x9cLatticexe2x80x9d types.
In the xe2x80x9cSunxe2x80x9d type, a polysaccharide is attached to a protein or peptide through a reactive group exclusively located at an extremity of the polysaccharide chain. Usually, this involves a carbonyl group located at the reductive end of the polysaccharide chain. Several polysaccharide chains may be attached onto the protein, the attachement usually involving an amino group carried by e.g., a lysine residue. Such conjugates are also defined as neoglycoconjugates. As a matter of example, a conjugate of this type is achieved in Alonso de Velasco et al, Infect. Immun. (1995) 63: 961, Paradiso et al, Vaccine Research (1993) 2 (4): 239, and Jennings U.S. Pat. No. 4,356,170.
In the xe2x80x9cRakexe2x80x9d type, peptides are attached along the polysaccharide chain. An example of this type is provided in Lett et al, Infect. Immun. (1994) 62: 785, and more appropriately, Lett et al, Infect. Immun. (1995) 63: 2645 and Kxc3x6nen-Waisman et al, J. Immunol. (1995): 5977. Attachement involves the amino group carried by the single lysine residue internal to the peptide sequence and/or the terminal amino group.
In the xe2x80x9cLatticexe2x80x9d type, the protein and the polysaccharide are cross-linked. This is made possible due to the fact that a protein rather than a peptide is used (usually amino or acid groups located along the protein) and that reactive groups located along the polysaccharide chain are involved. A conjugate of this type is described in Anderson U.S. Pat. No. 4,673,574. Schneerson et al, J. Exp. Med. (1980) 152: 361 also describes a conjugation method leading to a xe2x80x9cLatticexe2x80x9d typexe2x80x9d It uses CNBr and as a linker, adipic acid dihydrazide (ADH). Hydroxyl groups present all along the polysaccharide chain and side chain amino groups of the protein are involved.
Each of these structures may be achieved according to a variety of conjugation processes. The bound may be a direct bound as in Anderson U.S. Pat. No. 4,673,574, Jennings U.S. Pat. No. 4,356,170, Lett et al or Kxc3x6nen-Waisman et al. The bound may also be an indirect bound in that a linker molecule is used as illustrated by Schneerson et al. Additional to a linker, a spacer may also be used as described in Alonso de Valesco et al or Paradiso et al (for the pneumococcal polysaccharide). Various functional groups present on polysaccharide, protein, linker and optionally, spacer may be involved.
Some of the prior art reference cited above are presented with further details as follows:
In Alonso de Velasco et al, the carrier is a peptide of about 20 amino acid residues that comprises a single cysteine residue at either end. Streptococcus pneumoniae 17F polysaccharide is first derivatized at the reductive end by reductive amination with diaminopropane in the presence of NaCNBH3. Then the derivatized polysaccharide is bromoacetylated with N-succinimidyl bromoacetate as a linker and the polysaccharide so activated is coupled to the thiol group of the single N- or C-terminal cysteine residue of the peptide. A single-ended conjugate is thus obtained.
In Lett et al (1994), S. mutans or Saccaromyces cerevisiae polysaccharide are first oxidized with periodate so as to create aldehyde groups all along the polysaccharide chain. Then the oxidized polysaccharide is directly coupled by reductive amination to a peptide, in the presence of NaCNBH3.
In Paradiso et al, two polysaccharides are used: a pneumococcal polysaccharide and the polyribitol phosphate (PRP) of Haemophilus influenzae. Upon acidic hydrolysis of the pneumococcal polysaccharide, an aldose group is first created at the end of the polysaccharide chain. Amino groups are then added at the end of the chain upon reductive amination with diaminomethane in the presence of pyridine borane. The derivatized polysaccharide is activated with succinimidyl diester of adipic acid and coupled to amino groups of protein or peptide: Turning to PRP, this latter is first submitted to oxidative cleavage using periodate. Aldehyde groups are thus created at both ends. Oxidized PRP is then coupled to the amino groups of protein and peptide. In both cases, a xe2x80x9cSunxe2x80x9d structure is created.
In Kxc3x6nen-Waisman et al, the Vi polysaccharide and peptides (none of which contains a cysteine residue) or proteins are directly coupled together in the presence of (3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDAC):carboxyl groups of the polysaccharide and amino groups of the protein or peptides are involved in the conjugation. As a result, a xe2x80x9cLatticexe2x80x9d structure is created when the protein is used. When peptides are used, the structure depends upon the number of amino acid residue that bears amino groups. It may be either a simple xe2x80x9cLatticexe2x80x9d structure or a xe2x80x9cRakexe2x80x9d structure.
As may easily be understood, when conjugation involves functional groups present all along the polysaccharide chain, this leads to a cross-linked conjugate if the carrier is a protein (several amino or carboxyl groups on the protein are available for attachment). If the polypeptide carrier contains a single attachment site (this situation is very frequent when it is small enough), the conjugate thereof exhibits a xe2x80x9cRakexe2x80x9d structure (that may also be achieved if the polypeptide contains a few attachment sites and the conjugation carried out under close, uneasy control so that a single functional group on the polypeptide is reacted). When the conjugation method uses carboxyl or amino groups of a naturally-occurring polypeptide (or a fragment therof), this latter shall pretty small in order to get a xe2x80x9cRakexe2x80x9d structure, since carboxyl or amino groups are frequently encountered on polypeptides.
It has now been found a novel conjugation method that may easily produce a xe2x80x9cRakexe2x80x9d structure, while using the thiol group of a cysteine residue. Since cysteine residues are less frequent than lysine and aspartic acid, this method is therefore suitable for conjugating larger peptides.
As carrier, peptides have some advantages over proteins since they can easily be purified when biologically produced, or synthesized and therefore, are purer and more defined. Contrarily to carrier proteins which may also have detrimental properties (toxicity), peptides may be derived from those proteins so as to exhibit the carrier property only.
However, polysaccharide-peptide conjugates known in the art are less immunogenic than their polysaccharide-protein counterparts and definitively require adjuvantation. Surprisingly, polysaccharide-peptide conjugate made by the novel method have good immunogenicity. One of the reasons for this lies within the fact that the peptide moiety has a sufficient size.
Therefore, the present invention relates to a polysaccharide-peptide conjugate wherein the polysaccharide is advantageously immunogenic, which comprises:
(i) a peptide moiety having at least six amino acid residues, at least one of which being a cysteine residue;
(ii) a polysaccharide chain comprising at least four repeat units; and
(iii) a linker moiety bound to the thiol group of the cysteine residue and bound to (a) the native amino, hydroxyl or carboxyl groups of the polysaccharide chain or (b) amino groups created upon hydrolysis of the native N-acyl groups of the polysaccharide chain or (c) functional groups introduced on the polysaccharide chain upon derivatization with a spacer moiety bound to the native amino, hydroxyl or carboxyl groups of the polysaccharide chain.
Since the native amino, hydroxyl or carboxyl groups are found in the repeat units and therefore are present all along the polysaccharide chain, a conjugate of the invention typically exhibits a rake structure as described herein above. Accordingly, an alternative and equivalent definition for the conjugate of the invention may be provided as follows.
Said otherwise, a conjugate of the invention comprises a polysaccharide chain composed of repeat units and a plurality of peptide moieties, each moiety containing a cysteine residue and being covalently attached at random along the polysaccharide chain, through an indirect bound involving the thiol group of the cysteine residue and an amino, hydroxyl or carboxyl group of the polysaccharide, said indirect bound being achieved through either a linker or a spacer-linker moiety provided that the spacer entity of the spacer-linker moiety is linked to the amino, hydroxyl or carboxyl group of the polysaccharide.